Living cells are exposed to a variety of signals from their micro- and macro-environment. Signals are detected by receptors present on the cell surface and are then processed and transduced by biochemical cascades known as intracellular signaling pathways. Signal transduction through intracellular space is a key part of the cell communication and response and often involves the movement-or translocation of signaling proteins from one position to another within the cell. Novel methods for monitoring specific modulation of intracellular pathway in living cells could provide new opportunities in drug discovery, functional genomics, toxicology, etc.
Several diseases states can be attributed to altered activity of individual signaling components, such as protein kinases, protein phosphatases, transcription factors, etc. these signaling components are attractive as targets for therapeutic intervention. Protein kinases and phosphatases are well described components of several intracellular signaling pathways. Although the involvement of protein kinases in cellular in cellular signaling have been studied extensively, detailed knowledge of signaling-related translocation events needs convenient technology for its study.
Phosphorylation mediated by protein kinases is balanced by phosphatase activity. Translocation is observed within the family of phosphatases, and is likely to be indicative of phosphatase activity. See, e.g., Cossette et al., Exp. Cell Res., 223:459-66 (1996). Protein kinases often show a specific intracellular distribution before, during and after activation and monitoring the translocation processes of individual protein kinases or subunits thereof is thus likely to be indicative of their functional activity. Connection between translocation and catalytic activation has been shown for protein kinases like protein kinase C, cAMP-dependent protein kinase and mitogen-activated-protein kinase Erk-1. See, e.g., Debernardi et al., Proc. Natl. Acad. Sci. USA, 93:4577-82 (1996); Sano et al., Brain Res., 688:213-18 (1995).
Commonly used methods of detection of intracellular localization of protein kinases and phosphatases include immunoprecipitation, Western blots and immunocytochemistry. Translocation indicative of protein kinase C (PKC) activation has been monitored using different approaches such as immunocytochemistry where the localization of individual isoforms are detected following permeabilization of the cells; tagging all PKC isoforms with a fluorescent-label; chemical tagging of PKC with the fluorophore Cy3 and genetic tagging of PKCa and of PKCy and PKCe. See, e.g., Khalil et al., Am. Physiolog. Society, 263:C714 (1992); Godson et al., Biochim. et Biophys. Acta, 1313:69-71 (1996); Bastiaens et al., Proc. Natl. Acad. Sci. USA, 93:8407-612 (1996); Wagner et al., Exp. Cell Res., 258:204-14 (2000); Sakai et al., Soc. Neurosci., 22:371, Abstract 150.1 (1996).
Steroid receptors are hormone-dependent activators of gene expression. Steroid receptors mediate the action of steroid hormones (e.g., glucocorticoids, estrogens, progestins, testosterone, mineralocoticoids and 1,25-dihydroxycholecalciferol) in human tissues. After activation with the cognate ligand, receptors bind to chromatin in the nucleus and modulate the activity of target cellular genes. It is generally accepted that the unliganded glucocoticoid receptor (GR) resides in the cytoplasm, and that hormone activation leads both to nuclear accumulation and gene activation. See, e.g., Gasc et al., Steroid Hormone Receptors: Their Intracellular Localisation 233-50 (Clark, C. R., ed. Ellis Horwood Ltd. 1987; Beato, Cell, 56:335-44 (1989); Carson-Jurica et al, Endocr. Rev. 11:201-20 (1990); RONEMEYER, Steroid Hormone Action 94-117 (Parker, M. G., ed. Oxford University Press 1993); Tsai et al. Annu. Rev. Biochem. 63:451-86 (1994); Akner et al., J. Steroid Biochem. Mol. Biol. 52:1-16 (1995). However, the mechanisms involved in nuclear translocation and targeting of steroid receptors to regulatory sites in chromatin have been poorly understood. Green Fluorescent Protein has been used in an assay for the detection of translocation of the glucocorticoid receptor. See, e.g., Carey et al., Cell Biol., 133:985-96 (1996). Methods involving tagging a protein target with a luminophore (such as a fluorescent protein like GFP), expressing the luminophore-fusion protein in stably transfected cell lines, and quantifying the target movement in response to pharmacological stimuli by imaging is the subject of patents such as U.S. Pat. No. 6,518,021; EP 0986753B1; U.S. Pat. No. 6,172,188, and EP 0851874.
Directed protein movement in response to external stimuli is a mechanism employed by eukaryotic signal transduction pathways. Perhaps one of the best-studied in vivo signal transduction pathways is the NF-kB pathway, a convergent pathway for a number of different stimuli that impact the cell. Ligand binding and other stimulatory events at the cell surface trigger activation of the cascade that results in the eventual translocation of NF-kB from the cytoplasm to the nucleus. Proteins that are resident along a pathway offer a potential therapeutic targeting opportunity. Current technologies to track these events are limited to biochemical fractionation or fusion to fluorescent proteins.
Proteins have been labeled with fluorescent tags to detect their localization and conformational changes both in vitro and in intact cells. Such labeling is essential both for immunofluorescence and for fluorescence analog cytochemistry, in which the biochemistry and trafficking of proteins are monitored after microinjection into living cells. See, e.g., Wang et al., eds. METHODS CELL BIOL. 29 (1989). Traditionally, fluorescence labeling is done by purifying proteins and then covalently conjugating them to reactive derivatives of organic fluorophores. However, the stoichiometry and locations of dye attachment are often difficult to control, and careful repurification of the proteins is usually necessary.
Biochemical methods are often the most sensitive and quantitative however they are limited by their ability to discern sub-cellular structures without contamination from other organelles. In addition, the number of manipulations involved in preparing the samples makes these methods cumbersome and prone to high variability. The use of fluorescent proteins to track protein movement has positively impacted the scope and detail with which translocation events can be monitored. However the large amounts of protein necessary for efficient imaging make these experiments difficult to perform with toxic proteins and the supra-physiological levels of target protein can affect the quality of the data obtained. Further, the cell to cell variation is high, coupled to moderately low signal to noise ratios, making the assays more qualitative than quantitative.
Enzyme fragment complementation with beta-galactosidase (β-gal) was first shown in prokaryotes. See, e.g., Ullman et al., J. Mol. Biol. 24: 339-43 (1967); Ullman et al., J. Mol. Biol 32: 1-13 (1968); Ullman et al., J. Mol. Biol. 12: 918-23 (1965). Assays based on the complementation of enzyme fragments fused to interacting proteins that regenerate enzymatic activity upon dimerization are particularly well suited to monitor inducible protein interactions. Reviewed in Rossi et al., Trends Cell Biol. 10:1 19-22 (2000). These systems have important advantages including low level expression of the test proteins, generation of signal as a direct result of the interaction and enzymatic amplification. As a result, they are highly sensitive and physiologically relevant assays. See, e.eg., Blakely et al., Nat. Biotechnol. 18: 218-22 (2000). Additionally, assays based on enzyme complementation can be performed in any cell type of interest or in diverse cellular compartments such as nucleus, secretory vesicles or plasma membrane. The 6-galactosidase complementation system of U.S. Pat. No. 6,342,345 and as described in the literature enzymatically amplifies of the signal and can be used to monitor interactions in live cell in real-time. See, e.g., Rossi et al., Proc. Natl. Acad. Sci. USA 94: 8405-10 (1997); Blakely et al., Nat. Biotechnol. 18: 218-22 (2000).
Protein translocation is essential for mammalian cells to affect cellular responses, and convey information intracellularly. The use of GFP fusion proteins to track protein movement has revolutionized the ability to gather data regarding these actions and has been particularly useful in studying real-time kinetics of protein movement. However the difficulties associated with quantification of these events, such as small increases in fluorescence, high cell to cell variability, and the necessity for high expression levels of the fusion protein, prohibit its use in certain applications and limit the data to mainly qualitative measurements.
Therefore, what is desired is an assay combining both the localization aspects of fluorescence or luminescence-based assays, and the sensitivity and quantitative aspects of biochemical assays.